High-throughput screening (HTS) methods for identifying antagonists of chemoattractant receptors often rely on detecting perturbations in downstream events, such as cell migration. In the case of chemokine receptors, leukocyte cell migration is often assayed. However, compounds disrupting cell membranes or blocking downstream events mimic these outcomes, masquerading as candidate antagonists. Considerable efforts are then required to distinguish the genuine antagonists from those compounds or molecules that caused false positive signals. Identifying true antagonists, which represent only a small fraction of the large collections of candidate antagonists analyzed in high-throughput screens, is a formidable task. Realizing any savings in time or expense can bring a new drug to patients more quickly and less expensively.
Conventional assays that are adapted for use in HTS methods for screening small molecule antagonists of ligand-receptor interactions and signaling are usually one-dimensional. That is, they isolate and assay only the ligand-receptor interaction or the cellular signaling that ligand binding initiates, but not both. Because of this separation of physical interaction (ligand-receptor binding) from function (receptor signaling and downstream events), false positive signals are often observed, slowing discovery and development. False positives are molecules that give the desired result for undesirable reasons; they are often seen in screens for small molecule antagonists. Small molecules that initially appear to be inhibitors of receptor-ligand binding interactions (a desired result) may give such a result, for example, either by inhibiting the receptor-ligand interaction by binding the target receptor or ligand (desirable reasons), or by sickening or killing cells, or wielding other undefined effects (undesirable reasons).
Furthermore, conventional drug discovery formats for chemoattractant receptor antagonists fail to identify all clinically important molecules, a consequence of false negative signals. False negatives mean that clinically important molecules are undetected and remain undiscovered. For example, a conventional assay may identify a signal, as a result of binding of one or more molecules, i.e. a cluster of similar compounds. However, only the most potent molecule will be identified as a chemoattractant antagonist. As a consequence, a less potent molecule that permits chemoattractant receptor ligand-chemoattractant receptor binding, but inhibits chemoattractant receptor signaling, will be hidden in an initial screen for inhibitors of ligand binding.
One example of a conventional assay, the FLIPR® (Fluorometric Imaging Plate Reader) assay, illustrates these drawbacks. The FLIPR assay measures, over time, an intracellular mediator associated with activation of a cell bound receptor following exposure to a compound. Thus, FLIPR assays merely detect receptor-compound interactions that result in a change in the concentration of an intracellular mediator. The FLIPR assays may detect receptor-compound interactions that do not produce the downstream effect, some of which might also be considered false positives.
Chemokines, also known as “intercrines” and “SIS cytokines,” comprise a family of more than 50 small secreted proteins (e.g., 70-100 amino acids and about 8-10 kiloDaltons) which attract, activate, and act as molecular beacons for the recruitment, activation, and directed migration of leukocytes and thereby aid in the stimulation and regulation of the immune system, flagging pathogens and tumor masses for destruction. The name “chemokine” is derived from chemotactic cytokine, and refers to the ability of these proteins to stimulate chemotaxis of leukocytes. Indeed, chemokines may comprise the main attractants for inflammatory cells into pathological tissues. See generally, Baggiolini et al., Annu. Rev. Immunol, 15: 675-705 (1997); and Baggiolini et al., Advances in Immunology, 55:97-179 (1994).
There are two classes of chemokines, CXC (α) and CC (β), depending on whether the first two cysteines are separated by a single amino acid (C-X-C) or are adjacent (C-C). The α-chemokines, such as interleukin-8 (IL-8), neutrophil-activating protein-2 (NAP-2) and melanoma growth stimulatory activity protein (MGSA) are chemotactic primarily for neutrophils, whereas β-chemokines, such as RANTES, MIP-1α, MIP-1β, monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and eotaxin are chemotactic for macrophages, T-cells, eosinophils and basophils (Deng, et al., Nature, 381:661-666 (1996)).
The chemokines bind specific cell-surface receptors belonging to the family of G-protein-coupled seven-transmembrane-domain proteins (reviewed in Horuk, Trends Pharm. Sci., 15:159-165 (1994)), which are termed “chemokine receptors.” On binding their cognate ligands, chemokine receptors transduce an intracellular signal though the associated trimeric G protein, resulting in a rapid increase in intracellular calcium concentration.
While defending the individual from invading pathogens and tumors, an improper regulation of the immune system can result in a disease state. Inappropriate chemokine signaling can either promote infections (Forster et al., 1999) or lead to diseases associated with defective chemokine signaling, including asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, atherosclerosis (reviewed in Rossi and Zlotnick, 2000), graft rejection, and AIDS. Moreover, recent work has shown that particular chemokines may have multiple effects on tumors including promoting growth, angiogenesis, metastasis, and suppression of the immune response to cancer, while other chemokines inhibit tumor mediated angiogenesis and promote anti-tumor immune responses. Because chemokines play pivotal roles in inflammation and lymphocyte development, the ability to specifically manipulate their activity will have enormous impact on ameliorating and halting diseases that currently have no satisfactory treatment. Chemokine receptor antagonists can be used to obviate the generalized and complicating effects of costly immunosuppressive pharmaceuticals in transplant rejection (reviewed in DeVries et al., 1999).
One aspect of chemokine physiology that makes these proteins and their receptors especially attractive therapeutic targets is their specificity. Unlike cytokines, which have pleiotropic effects, chemokines target specific leukocyte subsets and, in some settings, attract these cells without activating them. Thus, antagonism of a single chemokine ligand or receptor should have a relatively specific outcome.
To expedite the identification of chemoattractant receptor antagonists, such as those for chemokine receptors, an assay that weeds out false signals by testing both chemoattractant receptor binding and a biological function would hasten drug development.